Modified nucleotide sequence encoding glucagon-like peptide-1 (glp-1), nucleic acid construct comprising same for production of glucagon-like peptide-1 (glp-1), human cells comprising said construct and insulin-producing constructs, and methods of use thereof

a nucleotide sequence and glucagon-like peptide technology, which is applied in the field of modified nucleotide sequence encoding glucagon-like peptide-1 (glp-1), can solve the problems of short half life, inability to rapidly degrade, and limited potential as a new therapeutic agent, so as to reduce increase the glucose blood level, and reduce the weight of the subj

Active Publication Date: 2008-12-04
BOEHRINGER INGELHEIM INT GMBH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0025]In another embodiment, there is provided a method of reducing weight in a subject, said method comprising: implanting into the subject an immunoisolatory device, wherein said device comprises isolated human cells stably transfected with (i) a recombinant expression vector comprising an isolated modified chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), a furin cleavable site between the human pro-insulin leader and the GLP-1(7-37), and a constitutive promoter which drives the expression of the nucleic acid sequence encoding GLP-1 constitutively, wherein the modified chimeric nucleic acid sequence is shown in SEQ ID NO:8, (ii) a second expression vector comprising an isolated nucleic acid sequence encoding human furin and a glucose-regulatable TGF-alpha promoter, wherein said promoter drives expression of the nucleotide sequence encoding human furin in a glucose-regulated manner, and (iii) a third expression vector comprising an isolated nucleic acid sequence encoding proinsulin, a furin cleavable site between the human pro-insulin leader sequence and insulin sequence and a constitutive promoter which drives the expression of the nucleic acid sequence encoding proinsulin constitutively; wherein the isolated stably transfected cells express a therapeutically effective amount of GLP-1 and insulin in a glucose-dependent manner upon stimulation with a concentration of glucose in the blood of the subject, wherein the stimulating concentration of glucose is higher than an ambient concentration of glucose, wherein the GLP-1 and insulin expression reduces the weight of the subject.
[0026]There is provided herein a method of reducing weight in a subject, said method comprising: wherein said device comprises isolated human cells stably transfected (i) a recombinant expression vector comprising an isolated chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), a furin cleavable site between the human pro-insulin leader and the GLP-1(7-37), and a constitutive promoter which drives the expression of the chimeric nucleic acid sequence constitutively, wherein the chimeric nucleic acid sequence is shown in SEQ ID NO:6, (ii) a second expression vector comprising an isolated nucleic acid sequence encoding human furin and a glucose-regulatable TGF-alpha promoter, wherein said promoter drives expression of the nucleotide sequence encoding human furin in a glucose-regulated manner, and (iii) a third expression vector comprising an isolated nucleic acid sequence encoding proinsulin, a furin cleavable site between the human pro-insulin leader sequence and insulin sequence and a constitutive promoter which drives the expression of the nucleic acid sequence encoding proinsulin constitutively; wherein the isolated stably transfected cells express a therapeutically effective amount of GLP-1 and insulin in a glucose-dependent manner upon stimulation with a concentration of glucose in the blood of the subject, wherein the stimulating concentration of glucose is higher than an ambient concentration of glucose, wherein the GLP-1 and insulin expression reduces the weight of the subject.
[0027]In another embodiment of the present invention, there are provided methods of treating a subject having Type II diabetes, treating a subject prone to hyperglycemia or suffering from hyperglycemia, and reducing weight in a subject, said methods comprising implanting into the subject an immunoisolatory device containing constitutive GLP-1 producing cells, as described below. The cells produce a therapeutically effective amount of GLP-1 constitutively.
[0030]Insulin production by pancreatic beta cells is thereby stimulated by the constitutively produced GLP-1 during an increase in glucose blood level in the patient. Advantageously, the methods of treatment may also include the implantation of engineered cells co-expressing furin and insulin, (in addition to implanted cells expressing GLP-1), which are also capable of producing insulin in glucose-regulated manner. The engineered cells insulin would provide an added therapeutic benefit for diabetic patients where endogenous insulin is not sufficient and also to cover the critical period until insulin producing pancreatic beta cells being responding to secreted GLP-1. The effect of the insulin production is reduction of the glucose blood level in the subject, thereby treating the Type II diabetes or the hyperglycemia.
[0031]In another embodiment, there is provided a method of reducing weight in a subject, said method comprising implanting into the subject an immunoisolatory device, wherein said device comprises isolated human cells stably transfected with (a) a recombinant expression vector comprising an isolated modified chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), a furin cleavable site between the human pro-insulin leader and the GLP-1(7-37), and a constitutive promoter which drives the expression of the nucleic acid sequence encoding GLP-1 constitutively, wherein the modified chimeric nucleic acid sequence is shown in SEQ ID NO:8 or (b) a recombinant expression vector comprising an isolated chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1)(7-37), a furin cleavable site between the human pro-insulin leader and the GLP-1(7-37), and a constitutive promoter which drives the expression of the chimeric nucleic acid sequence constitutively, wherein the chimeric nucleic acid sequence is shown in SEQ ID NO:6, wherein said cell produces endogenous furin. In a further embodiment, the GLP-1 is GLP-1 (7-36) rather than GLP-1 (7-37). The effect of the insulin production is a reduction in the weight of the subject.

Problems solved by technology

However, its potential as a new therapeutic agent is limited because this peptide cannot be administered orally, and it has short half life (about 5 minutes or less) in vivo.
However, because these molecules are foreign, they cannot be degraded rapidly in the body as native GLP-1.

Method used

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  • Modified nucleotide sequence encoding glucagon-like peptide-1 (glp-1), nucleic acid construct comprising same for production of glucagon-like peptide-1 (glp-1), human cells comprising said construct and insulin-producing constructs, and methods of use thereof
  • Modified nucleotide sequence encoding glucagon-like peptide-1 (glp-1), nucleic acid construct comprising same for production of glucagon-like peptide-1 (glp-1), human cells comprising said construct and insulin-producing constructs, and methods of use thereof
  • Modified nucleotide sequence encoding glucagon-like peptide-1 (glp-1), nucleic acid construct comprising same for production of glucagon-like peptide-1 (glp-1), human cells comprising said construct and insulin-producing constructs, and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

GLP-1 Constructs

[0116]In order to obtain a biologically active form of secreted GLP-1 from genetically engineered cells, the following strategy is used to generate the GLP-1 nucleic acid constructs. The active GLP-1(7-37) peptide is comprised of 31 residues, accordingly the nucleotide sequence corresponding to these residues is conveniently split in half to generate two separate fragments viz., N-terminal and C-terminal fragments as shown in FIG. 3-B. Complementary oligonucleotides corresponding to these amino acids are synthesized with the modifications listed below. Sequences of the oligonucleotides are listed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0117]An XhoI restriction site is added at the split site, without altering the amino acid sequence. A furin cleavage site (RQK′R) and the Kozak sequence and the bases consistent with the overhang of NheI restriction site are added to the 5′ end of the N-terminal fragment as shown in FIGS. 3-B and C. Bases consistent w...

example 2

Assessment of GLP-1 Secreted in Transiently Transfected Cells

[0123]Various human cell lines (293T, HeLa, SHP77, U2OS) are transiently transfected with GLP-1 constructs to assess production of GLP-1 in the culture medium. The transfections are performed as follows.

[0124]Cells are plated at 2.5×105 cells / 60 mm dish in 4.0 ml culture medium. The cells are allowed to adhere overnight. The cells are 40-60% confluent after the overnight culture. The medium is removed from the cells so that there is 1.5 ml remaining in the plate.

[0125]In a sterile microtube 1 μg DNA is added into a total of 150 μl DNA-condensation buffer (EC buffer, Qiagen Effectene transfection kit). Enhancer solution (8 μl l) is added into the DNA / EC buffer, vortexed for 1 second and incubated for 2-5 minutes at room temperature. 25 μl Effectene is added into each tube and vortexed for 10 seconds. The tubes are allowed to sit at room temperature for 5-10 min. The transfection mixture is diluted with 1.0 ml media and adde...

example 3

Engineering of Human U2OS Cells to Produce GLP-1

[0128]Human osteosarcoma cell line U2OS, available from the ATCC, is stably transfected with GLP-1 cDNA using the following protocol. The vector is linearized with BglII. The cells are co-transfected with pCDNA-huFurin expressing human furin. pCDNA-huFurin is linearized using MfeI. U2OS cells are seeded in 10-cm plates the day before at 2.5×105 per plate. The cells are transfected using calcium phosphate (Promega).

[0129]Three days post-transfection, medium is supplemented with 1000 μg / ml Geneticin. Once colonies are evident, cells are expanded in 6-well plates and tested for GLP-1 expression by active GLP-1 ELISA kit (Linco Diagnostic Services, Inc.). Cell populations producing high levels of GLP-1 are further cloned by limiting dilution at 1 cell / well. The clones are tested for their ability to produce GLP-1 in the presence and absence of DPPIV inhibitor.

[0130]The production of active GLP-1 in U2OS cells transiently transfected with n...

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Abstract

An isolated chimeric GLP-1 nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1 is provided. Also provided is an isolated modified chimeric GLP-1 nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1. Recombinant expression vectors comprising the chimeric GLP-1 nucleic acid sequences, which produce GLP-1 constitutively are provided, as are human cells transfected with such an expression vector in combination with an expression vector comprising a proinsulin nucleic acid sequence and an expression vector comprising a furin and a glucose-regulatable TGF-alpha promoter. Methods of producing human GLP-1 constitutively are provided as are method of producing GLP-1 and insulin or in a glucose-dependent manner using such transfected cells. Methods of treating a subject having Type II diabetes and methods of treating a subject prone to hyperglycemia or suffering from hyperglycemia are provided in which transfected cells produce human GLP-1 and insulin in a glucose-dependent manner. Also provided are methods of reducing weight in a subject by implanting into the subject transfected cells which produce human GLP-1 and insulin in a glucose-dependent manner.

Description

BACKGROUND OF THE INVENTION[0001]The present invention relates to nucleic acid constructs encoding human GLP-1 peptide and human cells engineered for expression of these nucleic acid constructs to produce GLP-1 peptide and insulin for the treatment and prevention of diabetes, hyperglycemia and weight reduction. The nucleic acid constructs comprise a natural or modified nucleotide sequence which encodes natural human glucagon-like peptide-1 (GLP-1). The modified construct was made using optimized codon usage (Codon Usage Database is available at www.kazusa.or.jp / codon / ) and nucleic acid fragment was synthesized commercially (Aptagen, Inc). Preferably, the constructs comprise natural or modified chimeric nucleic acid sequence encoding a human pro-insulin leader, a glucagon-like peptide-1 (GLP-1), and a furin cleavable site between the human pro-insulin leader sequence and the GLP-1. Engineered human cell lines comprising these constructs and which produce natural GLP-1 constitutively ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/00A61P43/00C07K14/00C12N15/00C12N15/11C12N5/06C12P21/04
CPCA61K38/00A61K48/00C07K14/495C07K14/605C07K14/62C07K2319/35C07K2319/50C12N15/85C12N2830/002A61P3/04A61P43/00A61P3/10
Inventor TATAKE, REVATI J.O'NEILL, MARGARET M.MONACO, KELLI-ANN
Owner BOEHRINGER INGELHEIM INT GMBH
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